1. Field of the Invention
This invention relates to an aromatic substituted glycoside, and more particularly to such a glydoside which serves as a substrate for the direct determination of alpha-amylase. The present invention also relates to an improved method for the synthesis of the substrates as well as related glycosides.
2. Discussion of the Prior Art
One of the most widely studied and accepted procedures in clinical chemistry is the determination of serum and urine alpha-amylase which is used for the diagnosis of pancreatic disease.
During the past twenty-five years various amylase methods have been developed for use in the clinical laboratory. Some of the methods, i.e. saccharogenic method, involve complicated methodology which makes their routine use prohibitive. Other methods, i.e., turbidometric and viscosimetric methods for the determination of alpha-amylase activity, are dependent on changes in the physical properties of the substrate which may be influenced to a considerable degree by other factors present in the serum. Today, one of the most widely used methods for alpha-amylase determination is the starch-iodine method. With this method only a specific portion of the substrate is measured and the enzyme does not work under substrate saturation conditions. Further, the presence of serum proteins could interfere with the starch-iodine reaction.
In addition to the above difficulties associated with the mentioned methods, a further difficulty is encountered because the aforementioned methods can be used to determine a rather limited range of alpha-amylase activity. Also, some of the methods cannot be used for accurate determination of either sub-normal or highly elevated alpha-amylase levels.
Synthetic substrates comprising nitro aromatic glycosides have been employed in alpha-amylase determinations, such as reported in U.S. Pat. No. 4,145,527. The alpha-amylase acts preferentially on the endo bonds to form smaller fragments and therefore in order to get complete action to generate the chromophore, e.g. nitrophenol, an additional supporting enzyme must be employed.
The use of aromatic glycosides directly without the use of an additional supporting enzyme has been reported but the results achieved therewith have proved to be impractical because of poor kinetics and/or poor rate of color release.
The assay involving a synthetic substrate is reported (Nature, 182 (1958) 525-526) in which a p-nitrophenol derivative of maltose is used. The p-nitrophenol replaces the anomeric hydroxyl group of maltose. Amylase causes cleavage of the substrate to produce p-nitrophenol which can be monitored at 410 nm. However, the assay is 16 hours long and maltase also cleaves the substrate. In this regard, Wallenfels, et al. , Carbohydrate Research, 61, 359 (1978), also reported the use of 4-(p)-nitrophenyl-alpha-maltotriose as a direct assay substrate for pancreatic alpha-amylase. In addition, Wallenfels, et al, Fresenius Z. Anal. Chem., 301, 169 (1980) , also reported the use of 2-(o)-nitrophenyl-alpha-maltotrioside. However these substrates also have proven impractical for use in a clinical assay for the reasons stated above. Wallenfels, et al. , supra 61, 359 (1978) also described the synthesis of a homologous mixture of p-nitrophenyl-alpha-maltooligosaccharides by enzymatic conversion employing alpha-cyclodextrtn as the glucosyl donor, p-nitrophenyl-alpha-glucoside as the glucosyl acceptor and Klebsiella pneumoniae cycloglucanotransferase as the enzymatic agent. Similarly, L. M. Hall, U.S. Pat. No. 4,225,672, reports the preparation of aromatic substituted-alpha-maltooligo-saccharides using cycloglucanotransferases from Bacillus strains. These methods yield mixtures of maltosides having typically 2 to 14 , and at times more, glucose units in the polymeric chain, and thus require laborious separation techniques to obtain the desired maltosides and maltotriosides, generally in poor yield. To reduce the complexity of the mixture of maltooligosaccharides to one containing primarily lower maltooligosaccharide, such as, alpha-maltosides, maltotriside, and maltotetraosides, trimming enzymes of the phosphorylase class affecting the non-reducing termini of the polymers having multiple glucose units, i.e., those having, for example, five or more such units, have been employed (see, for example, Wallenfels, et al., Offenlegungsschrift 2 752 501). It has now been found that it is more efficient and cost effective, to trim the higher maltosides with enzymes of the amylase class to reduce the number of compounds of the cycloglucanotransferase mixture to a manageable level and thereby facilitate separation of the desired lower maltooligosaccharides, particularly to the nitrophenyl- and chloronitrophenylmaltoside and maltotrioside.
The substrates and methods according to the present invention are distinguished from the known prior art by the combination that (1) additional supporting enzymes are not required to do the alpha-amylase analysis since the inventive substrates are acted upon by the alpha-amylase to directly cleave the substrate to generate the desired chromophore group; and (2) the kinetics are favorable, providing a useful rate of color release.
For example it has been found that 2-chloro-4-nitrophenyl-alpha-maltotrioside of the instant invention is hydrolyzed ten times faster than the prior art substrate, 4-nitrophenyl-alpha-maltotrioside under the optimum conditions for amylase assay. In addition, it has been found that the chromogens of the instant invention have improved spectral properties over monosubstituted nitrophenols in terms of higher molar extinction coefficients at the optimum pH for amylase assay. It has also been found that hydrazoic acid and alkali metal and alkaline earth salts thereof increase the rate of hydrolysis of the present maltooligosaccharides by alpha-amylase without effecting the molar extinction of the principal chromophore of the chromogenic moiety. Thus, sodium azide and lithium azide activate alpha-amylase and promote the enzyme induced hydrolysis of pancreatic and salivary alpha-amylase. Furthermore, the method of the present invention is simpler and more accurate than the methods of the prior art.